Abstract 1570: Macrophage mediated cell-kill evasion by human prostate cancer cells
Authors: Anniek Zaalberg, Emma Minnee, Wilbert Zwart, Andries M. Bergman
Journal: Cancer Research (2022)

Abstract Macrophages can adopt a cytotoxic M1 phenotype which can eliminate cancer cells. However, despite the presence of macrophages in the prostate, prostate cancer (PCa) is a prevalent cancer in men. This suggests that PCa cells can evade M1 macrophage mediated cell-kill. We have shown that human THP-1 derived M1-like macrophages readily kill human prostate cancer LNCap cells, while in testosterone proficient conditions (Fraction Growth over Mono-culture (FGM): 0.1). However, in testosterone deprived conditions LNCap cells survive (FGM: 1.1). In addition, when exposed to M1-like macrophage conditioned medium, LNCap cells are killed in testosterone proficient conditions and not in depleted conditions (FGM: 0.2 and 0.7, respectively). This suggests that M1 macrophage mediated PCa cell-kill depends on Androgen Receptor (AR) signaling and the release of soluble factors by macrophages. This is supported by cell-kill of castration-resistant LNCap-abl cells with an AR amplification (FGM: 0.1) in testosterone deprived conditions and no cell-kill in both testosterone deprived and proficient conditions of castration resistant PC3 cells without AR expression when co-cultured with M1-like macrophages (FGM: 0.7 and 0.7. respectively). To dissect the mechanism of M1 macrophage mediated cell-kill evasion, a genome wide CRISPR knock-out screen was performed in LNCap cells in co-culture with M1-like macrophages. LNCap cells were transfected with the Brunello library and cultured in testosterone proficient conditions. After 72 hours of co-culture, cells were harvested and DNA was isolated and submitted for sequencing. After quality check analysis, five gene knock-outs with a significant (log2FcMedian &amp;gt;1.5) survival advantage were identified, AR, PRKCD (Protein Kinase C Delta), IKBKA (Inhibitor of nuclear factor kappa-B kinase subunit α:), IKBKB (Inhibitor of nuclear factor kappa-B kinase subunit β) and IKBKG (Inhibitor of nuclear factor kappa-B kinase subunit γ). The latter three genes are components of the IkB kinase (IKK) complex, which is essential for activation of members of the nuclear factor-kB (NFkB) family of transcription factors. The hits as identified in the CRISPR knock-out screen, were validated in single guide knock-out LNCap cells in co-culture with M1-like macrophages. Additionally, we have shown that AR signaling enhances PRKCD expression, both at the RNA and protein level. Moreover, expression of PRKCD is significantly lower in prostatectomy specimen of 56 patients treated for 3 months with the AR signalling inhibitor enzalutamide prior to surgery, than untreated matched controls. Furthermore, LNCap cells lacking PRKCD, IKBKB and IKBKG were significantly less affected in their growth by reactive oxygen species (H2O2) than non-targeting LNCap cells. Ongoing studies are aimed to dissect how these critical factors are interconnected in LNCap cells, M1 macrophage mediated cell-kill evasion as a result. Citation Format: Anniek Zaalberg, Emma Minnee, Wilbert Zwart, Andries M. Bergman. Macrophage mediated cell-kill evasion by human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1570.